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3 - Experiments in growth chambers

3 - Experiments in growth chambers

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1.4 Hydroponics

FIGURE 1.2  Climate-Controlled Growth Chamber.

2. Although growth chambers enable a strong temporal control over conditions,

spatial variability is often larger than anticipated and higher than those

measured in experimental fields. For example, light intensity may vary from

place to place in the growth chamber (Granier et al., 2006) and can be especially

lower close to the walls.

3. Gradients in air velocity may go unnoticed in growth chambers, although they

can affect evaporative demand. Variation in air circulation may be especially

large when plant density is high or plants are placed in trays, which may block

air circulation around the plants. Both too high and too low wind speeds are


4. A factor that may strongly vary in a temporal manner is the local atmospheric

CO2 concentration; generally, CO2 levels in a building are higher than outside.

5. Under greenhouses as well as growth chambers crops are experimented through

either hydroponics or pot culture method of growing crops.


Roots provide nearly all the water and nutrients that a plant requires. If the aim is

to design an experiment in which these two factors have the least limiting effect on

growth, then hydroponics or aeroponics is the preferred choice (Gorbe and Calatayud, 2010). Hydroponics systems can be either based on roots suspended in a water



CHAPTER 1  Various methods of conducting crop experiments

FIGURE 1.3  Hydroponics Experiment.

solution or in some solid medium such as sand, rockwool, or another relatively inert

medium, which is continuously replenished with nutrient solution (Cooper, 1979).

Frequently used nutrient solutions were described by Hoagland and Snijder (1933)

and Hewitt (1966), although the truly optimal composition is species specific. Preparation of macro, micronutrients (Appendix VI) and Hoagland solution (Appendix

VII) were given in appendices as ready recoknoire. Hydroponics experiment is

shown in Fig. 1.3.


1. Water-based systems have the advantage that they allow easy experimental

access to the roots for physiological or biomass measurements. However, care

has to be taken while roots are transferred from one solution to another, as

breakage of roots may easily occur.

2. There is also a need to take into account the composition of tap water when

setting for the final composition. Because of the much higher mixing rate

in soilless systems and the direct access of plant roots to the nutrients, the

concentrations of nutrients that are needed to sustain supply are 5–10 times

lower than those required for plants growing on sand where there is an absence

of continuous flow through.

1.5 Pot culture

3. Ensure that the concentration of macro and especially micronutrients in a

hydroponics system is not too high, as this will negatively affect plant growth

or may even cause leaf senescence (Munns and James, 2003). On the other

hand, nutrient concentrations should not become too low either, as plants will

otherwise deplete the available minerals. Hence, regular replacement of nutrient

solution is necessary.

4. Bigger plants usually need more nutrients and so the rate of replenishment must

increase with plant size, unless the nutrient concentration itself is continuously

monitored and adjusted.

5. Good mixing of aerated nutrient solution is vital to avoid depletion zones

around the roots and anaerobic patches, but should not be too vigorous to avoid

strong mechanical strains. In addition, specific uptake mechanisms such as the

release of chelating agents to increase iron availability (Romheld, 1991) or the

release of organic acids by the root may be affected.

6. The pH of the hydroponic solution may increase or decrease, depending

on whether nitrate or ammonium is present in the solution and the specific

preference of a given species. For most plant species a pH of 6 seems to be

optimal, although specific species may deviate significantly. Monitoring and

adjusting the pH of the solution at a regular basis is highly recommended,

keeping in mind that pH changes are stronger in small volumes of nutrient

solution and for roots with faster nitrogen uptake rates.

7. It should also be checked that there is no accumulation of salts at the root: shoot

junction over time, as this can damage the seedlings of some plant species.


An alternative to hydroponics is to grow plants in pots filled with an inert solid

medium (eg, sand, perlite) or soil and to water them regularly or on demand. Use

of pots with a solid substrate may at least mimic the higher mechanical impedance

to root growth that plants experience in soils and allows for a higher homogeneity

and control of the nutrient and water conditions than in soil. Pot culture (Fig. 1.4)

allows more freedom in the choice of the location of the experiment and ensures

FIGURE 1.4  Pot Culture Experiment.



CHAPTER 1  Various methods of conducting crop experiments

easy handling and manipulation of the shoots of individual plants. Most overlooked

factors in pot culture are pot size and the fact that nutrients and water supply strongly

interact with plant size.

1. Pot size: The size of the rooting volume also requires careful attention. The

smaller the pot, the more plants fit into a growth chamber or glasshouse, an

advantage for nearly all laboratories where demand for space is high. At the

same time, in most experiments smaller pots will also imply a lower availability

of below-ground resources and if pots are closely spaced, also a comparatively

lower amount of irradiance available for each plant. Moreover, the smaller the

pot the stronger roots become pot-bound, leading to undesirable secondary

effects. In experiments in which rooting volume varies, there is almost

invariably a strong positive correlation between plant growth and pot volume

reported. Conditions obviously differ from experiment to experiment, but as

a rule of thumb, pot size is certainly small if the total plant dry mass per unit

rooting volume exceeds 2 g/L (Poorter et al., 2012).

2. Precautions:

a. Demands for water and nutrients increase strongly with the size of the

plants, so the water and nutrient availability that are amply sufficient for

small plants at an early phase may become limiting at later developmental


b. Nutrient availability of commercially provided soil will vary among

suppliers and even over time from soil batch to soil batch. Mixing of slowrelease fertilizer with the soil or regular addition of nutrient solution may

mitigate this problem to some extent.

c. Root damage may occur if pots are black and warm up under direct solar

radiation. Moreover, soil temperature per se and even gradients in soil

temperature within single pots can affect plant growth and allocation

(Fullner et al., 2012).

Phenotyping experiments with plants require careful planning. The most controlled growth environment is not necessarily always the best one. Growing crop

plants for experimental purposes remains an art, requiring in-depth knowledge of

physiological responses to the environment together with proper gauging of environmental parameters. Hence, it is advocated to adopt a practical checklist (Table 1.1)

to document and report an asset of information concerning the abiotic environment,

plants experienced during experiments. Similarly, advantages and disadvantages of

field versus controlled environments in relation to some physiological traits are given

in Table 1.2.

1.5 Pot culture

Table 1.1  Checklist With the Recommended Basic and Additional Data to

Be Collected in All Methods of Experimentation

Sr. No.

Basic Data

Additional Data

1. Light intensity


• Average daily integrated

PPFD measured at plant or

canopy level (mol m−2 day−1)

• Average length of the light

period (h)

2. Light quality

• For GC and GH: type of

lamps used

3. CO2

• For GC and GH: controlled/


• Water-based hydroponics/

solid-based hydroponics

including substrate used/soil


• Container volume (L)

• Number of plants per


• For hydroponics and soil: pH

• Frequency and volume of

replenishment or addition

• For hydroponics: composition

• For soil: total extractable N

before fertilizer added

• For soil: type and amount of

fertilizer added per container

or m2

• Average VPD air during the

light period (kPa) or average

humidity during the light

period (%)

• For pots: volume (L) and

frequency of water added per

container or m2

• Average day and night

­temperature (˚C)

• For GC: light intensity (m mol

m−2 s−1)

• Range in peak light intensity

(m mol m−2 s−1)

• For GH: fraction of outside

light intercepted by growth

facility components and

surrounding structures

• R/FR ratio (mol mol−1)

• Daily UV-B radiation (W m−2)

• Total daily irradiance (W m−2)

• Average [CO2] during the light

and dark period (m mol mol−1)

• Container height

• For soil: soil penetration

strength (Pam−2); water

retention capacity (g g−1 dry

weight); organic matter

­content (%); porosity (%)

• Rooting medium temperature

4. Rooting medium

5. Nutrients

6. Air humidity

7. Water supply

8. Salinity

• Composition of nutrient

­solutions used for irrigation

GC, growth chamber; GH, glass house.

Adapted from Poorter et al. (2012).

• For soil: concentration of P

and other nutrients before

start of the experiment

• For soil: total extractable N

at the end of the experiment

• Average VPD air during the

night (kPa) or average

humidity during the night (%)

• For soil: range in water

­potential (MPa)

• For soil: irrigation from top/

bottom/drip irrigation

• Changes over the course of

the experiment

• For hydroponics: composition

of the salts (mol L−1)

• For soils and hydroponics:

electrical conductivity (dS m−1)




Controlled Facilities

Traits to Study







Less uniform

Dependence on



Control of the intensity,

uniformity, timing, and

repeatability of treatments.

Out-of-season experiments

are possible

Interactions between ­factors

can be controlled

Particular variables

(­radiation, ozone, etc.)

can be manipulated and


Control of environmental



Control of water applied

Confounded by plant

growth rate and differences

in water status

Pot experiment limitations

on root growth

Control of root depth

Equal soil water potential by

growing all genotypes in the

same pot

Unrealistic (rapid) drying/

rehydration cycles

Unpredicted interactions

Responses to drought

Realistic drying cycles

Realistic interactions with

environmental factors

Realistic soil profile for

root development

Osmotic adjustment

Cooccurrence of

­additional stresses (heat,

low temperature)

Less control over


Confounding factors

(toxicities, salinity)

Confounded by root

depth and differences in

soil water potential

Variation in the glasshouse

environment and handling

of materials

Unrealistic (rapid)

drying cycles

CHAPTER 1  Various methods of conducting crop experiments

Table 1.2  Advantages and Disadvantages of Field Versus Controlled Environments in Relation to Some Physiological Traits


Traits to Study


Transpiration efficiency

Canopy temperature

Integrative measurement,

scoring the entire canopy

of many plants Related to

the capacity of the plants

to extract water from

deeper soil profiles

Root growth studies

Realistic soil profile

(Biomass, length,

growth rate, etc.)

Adaptation to harsh



Transgenic plants



Controlled Facilities



Water fluxes cannot be


Measurements must be

taken when the sky is

clear and there is little or

no wind

Precise control of water



Complete root systems are


High sampling variance

Uniform sampling

Soil properties difficult

to manipulate

Risk of pollen flow

Strict regulations and


Soil properties can be



Low risk of pollen flow

Less/easier regulations

Pot experiment limitations

Control of external factors


Only single plant/small


of plants can be screened

Not related to the capacity

to extract water from

deeper soil profiles -unless

special pots are used

Pot size, temperature,

salinity, and hypoxia limiting

root growth

Adapted from Reynolds et al. (2012).

1.5 Pot culture


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2 Seed physiological and biochemical traits . . . . . . . . . . . . . . . . . . . . . . . . 17

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Seed physiological

and biochemical traits


Seed is the basic input in agriculture. It differs from other inputs in terms of having

life. Hence, scientific methods are involved in producing and storing it. Maintenance

of seed quality is mandatory in selling seed lots. Seed lots are evaluated on the basis

of their germination capabilities and vigor. Both germination and vigor of a plant

depend on the environment to which plant is exposed, especially from grain filling

stage. Genotypic variability in vigor and initial seedling establishment was noticed

among crop genotypes. Hence, several physiological and biochemical methods of

evaluating crop seed for viability and vigor are described in this chapter.



Seed viability is the ability of seed to germinate and produce “normal” seedlings.

In another sense, viability denotes the degree to which a seed is alive, metabolically

active, and possesses enzymes capable of catalyzing metabolic reactions needed for

germination and seedling growth.  Seed viability tests

1. Tetrazolium test: This test is often referred to as quick test since it can be

completed within hours. The test is usually based on measuring the activity of

dehydrogenase enzyme in the tissues of embryo. It is conducted by using 2, 3,

5-triphenyl tetrazolium chloride (TTC) solution.


Any living tissue must respire. In the process of respiration the enzyme dehydrogenase will be in a highly reduced state. When the seed is treated with the colorless

tetrazolium solution, the living tissue of the seed by virtue of respiration and having the dehydrogenase enzyme in a highly reduced state gives off hydrogen ions.

These hydrogen ions reduce the colorless tetrazolium solution into red colored

formazan. Thus, the tetrazolium test distinguishes between viable and dead tissues

of the embryo on the basis of their relative rate of respiration in hydrated state.

2, 3, 5 - Triphenyl tetrazolium chloride → Triphenyl formazan + HCl


(red color)

oxidized state

reduced state

Phenotyping Crop Plants for Physiological and Biochemical Traits. http://dx.doi.org/10.1016/B978-0-12-804073-7.00002-8

Copyright © 2016 BSP Books Pvt. Ltd. Published by Elsevier Inc. All rights reserved.


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